Substance Use Disorders (SUD) involve changes in dopamine and glutamate transmission which imply non-adaptive plastic changes in cortico-striatal circuitry. Our previous studies have demonstrated a key role of adenosine in the modulation of striatal dopamine and glutamate transmission, largely mediated by G protein-coupled receptor (GPCR) heteromers, functional complexes of different adenosine and dopamine receptor subtypes. At the postsynaptic level, adenosine A1 and A2A receptors (A1R and A2AR) are co-segregated with dopamine D1 and D2 receptors (D1R and D2R) in the GABAergic striato-nigral and striato-pallidal neurons, respectively, forming A1R-D1R and A2AR-D2R heteromers. adenosine and dopamine receptors are also localized at the presynaptic level, in the cortico-striatal terminals, but forming heteromers of either adenosine or dopamine receptor subtypes, mainly A1R-A2AR and D2R-D4R heteromers, which exert a significant control of glutamate release. Our research project deals with the study of the functional and pharmacological properties of these striatal presynaptic and postsynaptic GPCR heteromers, and their role in SUD and other neuropsychiatric disorders that involve dysfunctional dopamine and glutamate transmission (Parkinsons disease, L-dopa-induced dyskinesia, Huntington disease HD, schizophrenia, attention deficit hyperactivity disorder or ADHD and Restless Legs Syndrome or RLS), and their possible role as targets for the treatment of these neuropsychiatric disorders. Furthermore, we are also exploring the possibility of GPCR heteromers localized in the ventral tegmental area (VTA) that modulate dopamine cell function and, therefore, could also constitute targets for SUD and for the treatment of loss of control of food intake. Our more recent studies on the A2AR-D2R heteromer have provided a significant conceptual discovery in the field of GPCR pharmacology. Using a peptide-based approach, we obtained evidence for the existence of functional pre-coupled complexes of heteromers of A2AR and D2R homodimers (A2AR-D2R heterotetramer) coupled to their cognate Gs and Gi proteins and to subtype 5 adenylyl-cyclase (AC5) (1). We also demonstrated that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled receptor (D2R) to counteract AC activation mediated by a Gs-coupled receptor (A2AR) (1). The results of this study, which involved mammalian transfected cells and striatal cells in culture, strongly suggested that A2AR-D2R-AC5 complexes should constitute a main functional population of both striatal A2AR and D2R (1). With the addition of new behavioral experiments on A2AR- and D2R-deficient mice with the same genetic background (2), we could then readdress the results of previously published studies on the behavioral effects of pharmacological and genetic blockade of A2AR and D2R in the frame of one predominant population of striatal A2AR and D2R forming A2AR-D2R-AC5 complexes (3). Using similar molecular tools (synthetic peptides) than those used to interrogate the molecular structure of the A2AR-D2R heteromers, we have also been able to confirm that the A1R-D1R heteromer conforms to the same basic heterotetrameric structure and that the canonical antagonistic interaction at the AC level by which a Gi-coupled receptor (A1R) counteracts AC activation mediated by a Gs-coupled receptor (D1R), is a functional property of receptor heteromers (4). In addition, we found a new significant localization of the A1R-D1R in the central nervous system, the spinal motoneuron. With the in situ application of peptides on rat spinal cord tissue, we could demonstrate that the A1R-D1R heteromer exerts a very significant control of the excitability of the spinal motoneuron, which mediates the spinal locomotor activation effects of caffeine (4). On the other hand, using similar molecular tools (synthetic peptides), we found a different quaternary structure for the also Gi-Gs-coupled A1R-A2AR heterotetramer (compact rhombus-shaped, instead of the open linear arrangement of the A2AR-D2R heterotetramer), which did not allow a canonical Gs-Gi interactions at the AC level (5). This provides the A1R-A2AR heteromer with a specific G protein-dependent inter-communication by which A2AR activation does not allow A1R to signal through its coupled Gi protein (5). This mechanism provides the A1R-A2AR heteromer with is ability to serve as an adenosine concentration-dependent switch that controls cortico-striatal glutamate release. We have postulated that alterations in the quaternary structure of the A1R-A2AR heteromer can be involved in the recently reported association of an A1R gene (ADORA1) polymorphism and early-onset Parkinsons disease (6). But of more societal significance, due to its prevalence, we have provided evidence that indicates that alterations in the stoichiometry of A1R and A2AR in the cortico-striatal terminals represent a key pathogenetic mechanism involved in the akathisia and periodic leg movements during sleep in restless legs syndrome (RLS). We have previously demonstrated a significant downregulation of A1R and up-regulation of A2AR in the rodent with brain iron deficiency (BID), a well-accepted pathogenetic model of RLS. Uing optogenetic-microdialysis techniques, we demonstrated that BID in rodents is associated with a hypersensitivity of cortico-striatal terminals to release glutamate (7). Our working hypothesis is that RLS is associated with a hypoadenosinergic state that secondarily drives hyper- and hyperdopaminergic states, which determine RLS symptomatology (8, 9). We have also demonstrated that the drugs that are clinically effective in RLS are targeting the hypersensitive cortico-striatal glutamatergic terminals and, more specifically, the D2R-D4R heteromer (pramipexole an ropinirole) and the 2 subunit-containing voltage dependent calcium channels (2 ligands, such as gabapentin). The next obvious step was targeting the A1R in the A1R-A2AR heteromer, which was achieved by using dipyridamole, an inhibitor of the adenosine transporter ENT1 (9). We have then provided preliminary clinical evidence (an open trial) that dipyridamole or other EMT1 blockers can constitute a new therapeutic approach for RLS (10). Finally, we have exploited a series of biophysical techniques, based on bioluminescence resonance energy transfer (BRET) and bimolecular luminescence complementation, to provide reliable measurements of ligand-induced changes in the interactions between the receptor and the different subtypes of G proteins; more specifically, the G stimulatory subtypes Gs and Golf, and the G inhibitory subtypes Gi1, Gi2, Gi3, Go1 and Go2 (11-13). These methods have allowed us to demonstrate the existence of G protein subtype-dependent functional selectivity (14, 15). For instance, the D1R agonist dihydrexidine is a full agonist with Gs, but a low efficacious partial agonist with Golf, which endow this ligand with a preferential cortical versus striatal selectivity, therefore with possible implications for treating the negative cognitive symptoms of schizophrenia. We are now analyzing this type of functional selectivity in the frame of GPCR heteromerization.